Many of the low molecular weight drugs used in chemotherapy rapidly enter all types of cells by random diffusion through the cell membrane. This lack of selectivity decreases their availability at the desired target tissue and sometimes causes undesirable side effects. Cellular uptake is rapid so that the therapeutic effect is not extended over a period of time. Furthermore, glomerular filtration can rapidly remove the drugs from the bloodstream.
The covalent attachment of low molecular weight bioactive molecules to soluble polymeric carriers prevents both glomerular filtration and cellular absorption by simple diffusion. Uptake is restricted to cells capable of a substrate selective mechanism known as pinocytosis, in which a region of the limiting membrane of the cell engulfs the macromolecule and is then detached inwards to form a free intracellular vesicle containing the captured material.
This difference in uptake mechanisms affords a potential method for directing drugs specifically to those cells where their therapeutic effect is required.
A further difference lies in the subsequent fates of the two types of molecule i.e, polymer bound or free drug. Small molecules which enter by diffusion tend to find their way to all parts of the cell, but macromolecules, following pinocytosis, are transported in their intracellular vesicles directly to the lysosomal compartment of the cell where an array of hydrolytic enzymes is available.
The pinocytic uptake of a polymeric drug in which the drug-carrier linkage is susceptible to lysosomal hydrolysis therefore affords a mechanism for the controlled intracellular release of a bioactive molecule leading to its appearance within the cytoplasm of the target cell. The theoretical considerations involved in the design of such a drug system have recently been reviewed in an article by J. Kopecek entitled "Synthesis of Tailor-made Soluble Polymeric Carriers" in Recent Advances in Drug Delivery Systems (Plenum Press, 1984).
In order to design such a system, two criteria must be satisfied. First, a drug-carrier linkage must be devised which undergoes controlled lysosomal hydrolysis, but is capable of withstanding the action of enzymes in the bloodstream. Second, the drug delivery system must be able to achieve specific uptake at those target cells where the therapeutic effect is required, with minimal uptake by other cells.
There are basically three forms of pinocytosis: fluid phase, adsorptive, and receptor-mediated. Fluid phase pinocytosis is the most general form in which soluble macromolecules and solutes enter the cell in liquid droplets. Many, if not all nucleated cells use fluid phase pinocytosis to internalize material from the extracellular space. It is known as a "constitutive" process because it is continuous (as opposed to triggered as is phagocytosis) in that the cell is always ingesting pieces of its plasma membrane.
Adsorptive pinocytosis is also a relatively indiscriminate process. However, in this case a macromolecule may physically adsorb (nonspecifically) to a site on the cell membrane, and then by the invagination process be taken in by the cell.
Receptor-mediated pinocytosis is by far the most specific form of pinocytosis by which a macromolecule with a marker complementary to a cell surface receptor binds to that receptor and is subsequently internalized to the cell interior. In this way, macromolecules such as hormones, transport proteins, proteins modified for degradation, growth factors, and some antibodies are taken in by cells from the extracellular fluid. The advantage of receptor-mediated pinocytosis lies in the fact that a higher concentration of ligand may be internalized in specific cells than by the other mechanism.
Regardless of the mode, once internalized by pinocytosis, the ultimate leading fate of a solute is delivery to secondary lysosomes where it can be degraded and distributed by cell in various ways. Naturally, since during the process of fluid phase pinocytosis there is indiscriminate uptake of cell surface markers, the cell is equipped with the machinery to recycle essential lipids and proteins back to the cell membrane.
Although the process of pinocytosis affords a degree of selectivity towards macromoleculcs, a selectivity which can be optimized, e.g., by varying the molecular weight, greater target selectivity can be achieved by the incorporation within the macromolecule of a specific "targeting moiety". Cells possess specific receptors and cell antigens on their surfaces which "recognize" and interact with certain types of molecular entities known as specific determinants. High cell specificity can be achieved by the incorporation in the polymeric drug of a determinant which is recognized by the type of cells in which the therapeutic effect is required.
Thus, a drug delivery system which would allow specific targeting followed by intracellular drug release requires the following features:
(a) an inert polymeric carrier, which is preferably susceptible to lysosomal hydrolysis to facilitate elimination of the polymer from the body, PA1 (b) a degradable drug-carrier linkage which is resistant to extracellular hydrolysis, but which is subject to controlled lysosomal hydrolysis, and PA1 (c) an optional targeting moiety if desired.
Although natural macromolecules have been used as carriers, synthetic polymers offer the advantages that the molecular weight can be more readily adjusted for optimum cell selectivity and, unlike many natural macromolecules, they are not immunogenic. They also lend themselves more readily to commercial production.
Synthetic polymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) have been proposed as potential drug carriers, see U.S. Pat. Nos. 4,062,831 and 4,097,470; such polymers are soluble in aqueous media and have good biocompatibility. Furthermore, by the incorporation of p-nitrophenylesters of N-methacryloyl oligopeptides they can be combined with many drugs which contain a primary amino group. The polymeric chains may be cross-linked to a level below the gel point in order to achieve the optimum molecular weight and to provide, by the use of biodegradable cross-linkages, a means of degrading the polymer to facilitate elimination from the body.
Since lysosomal enzymes include a number of proteinases with the ability to hydrolyse peptide linkages, direct linkage of the bioactive molecule to the polymer chain by an amide bond would appear to have the potential for lysosomal hydrolysis. In practice, this is not found to be the case. However, peptide "spacers" interposed between the drug and the carrier have been found to undergo degradation by lysosomal enzymes within a broad range of rates. The bond actually cleaved is usually that between the drug and the neighboring amino acid, although this is not always the case. The rate of hydrolysis, that is the rate of drug release, is found to depend greatly on the number and the nature of the amino acid residues in the peptide spacer. Spacers of less than two amino acids are not generally susceptible to lysosomal hydrolysis. Peptide spacers designed to match the known substrate specificity of thiol-proteinases, known to be present in lysosomes, are particularly effectively cleaved.
It has been demonstrated that the modification of glycoproteins to give oligosaccharide side-chains which terminate in galactose leads to a dramatic increase in the deposition of the glycoproteins in the parenchymal cells of the liver. The galactose moiety acts as a specific determinant interacting with receptors localized on the plasma membrane of the liver cells. This offers a potential mechanism for the targeting of drugs to hepatoma, a particularly difficult cancer to treat. Furthermore, galactosamine bound to HPMA copolymers by an amide bond gives a similar result, indicating that receptors on hepatocyte membranes recognize the galactose moiety not only in glycosides, but also when present as N-acyl galactosamine. A number of other recognition systems are known, for example, the N-acetylglucosamine/mannose recognition system of Kupffer cells and macrophages and the phosphohexose recognition system of fibroblasts.
Another possible targeting mechanism is to bind the polymeric drug to an antibody which is recognized specifically by those cells which have the appropriate antigenic receptors. Drug molecules have been bound directly to immunoglobulins, but this can lead to loss of drug activity, loss of antibody activity and/or solubility of the conjugate.
A further targeting mechanism is to include a protein or a hormone, for example transferrin and melanocyte-stimulating hormone, which will bind specifically to the target cell type.
While the desirability of synthesizing targeted polymeric drugs with hydrolyzable peptide spacers has been referred to in the prior art (see Kopecek, supra), the identification of peptide spacers which are capable of controlled intracellular drug release at a satisfactory rate and the identification of linking group/determinant combinations which give good targeting to the desired cell receptors, are matters of continuing research.
As discussed earlier, the rate of lysosomal hydrolysis of a peptide spacer is dependent on both the number and the nature of the amino acid residues. This is a reflection of both steric and structural factors. Thus the rate of terminal hydrolysis of a spacer containing 2 to 4 amino acid residues is generally dependent on the number of residues present, an effect attributed to stearic interaction between the polymer chain and the enzyme.
For a given length of peptide, the rate of hydrolysis is dependent on the nature (and sequence) of the amino acid residues. This dependency arises from the substrate specific nature of the lysosomal enzymes responsible for cleavage of the peptide spacer. The region of the enzyme where interaction with the substrate takes place is known as the "active site" of the enzyme. The active site performs the dual role of binding the substrate while catalyzing the reaction, for example cleavage. Studies of the structures of the complexes of proteolytic enzymes with peptides indicate that the active site of these enzymes is relatively large and binds to several amino acid residues in the peptide.
Thus the degradability of a particular bond in a peptide chain depends not only on the nature of the structure near the cleaved bond, but also on the nature of the amino acid residues which are relatively remote from the cleaved bond, but play an important part in holding the enzyme in position during hydrolysis. So far the detailed structures of the active sites of lysosomal enzymes have not been determined and this has proved to be an obstacle to the preparation of peptide spacers which undergo lysosomal hydrolysis at a suitable rate for use in polymer drugs.